Effects Of Energy Metabolism During Mouse Oocyte Aging And The Study On The Effects Of Male On The Development Of Mouse Embryo

Chapterâ… : Effects of energy metabolism during mouse oocyte agingmetaphase II (MII)-arrested oocyte undergone oocyte aging afer delay in oviduct or culture media. Fertilized aged oocytes usually result in developmental anomaly. In addition,oocyte aging impact in vitro oocyte operation ,such as in vitro fertilization and nuclear transplantation and so on. In human,another insemination is needed if the first insemination failed. In vitro culture system may influence the second insemination, so it is necessary to investigate the underlying mechanism of oocyte aging. The previous reports demonstrated MPF activation decreased during oocyte aging, which lead to increasing of the sensitiveness to parthenogenetic activation and rate of activation. The rate of activation can reflect the degree of oocyte aging, so we detected oocyte aging by observing the rate of oocyte activation. We studied energy metabolism during oocyte aging by supplying glucose, sodium lactate , sodium pyruvate and its specific inhibitors of metabolism pathway. By this way, we can provide proof for regulating oocyte aging by altering energy substance, which is very important to optimize in vitro culture condition of oocytes. The results are summarized as follows:1. The oocytes cultured in CZB+FCS/BSA/PVA undergone similar oocyte aging. DO in these medias didn't undergo aging, but more than half COC did and the activation rate is significant higher in M199 than in CZB. COC in M199±PVA were obviously different from in above groups. DO undergone rapid oocyte aging, however COC didn't.2. Both DO and COC undergone rapid oocyte aging afer being cultured in CZB without glucose, sodium pyruvate and sodium lactate, and the activation rates are 93.3% and 91.8% respectively, but neither DO nor COC undergone oocyte aging in meida supplied with either sodium pyruvate or sodium lactate alone, the activation rates were all lower than 5%; DO undergone rapid oocyte aging,with 95.3% activation rate, however COC didn't ,with 0 activation rate, in CZB supplied with glucose alone; Few oocytes undergone aging in CZB supplied with three or two substance.3. The activation rate of COC cultured in CZB containing glucose supplied with 100μM or 200μM DHEA, inhibitor of PPP path, were all lower than 5%.4. The activation rate gradually increased when increasing the concentration ofoxamate, inhibitor of LDH, in CZB containing sodium lactate. Activation rates were49.2% and 59.6% respectively. But activation rate of oocytes cultured in CZBcontaining sodium pyruvate supplied with oxamate, remained unchanged.5.Activation rate gradually increased when the oocytes were cultured in CZBcontaining sodium pyruvate with increasing concentration of rotenone. When theconcentration of Rotenone were 0.05μM,30% oocytes were activated,while thecongcentration were 0.1μM, 84% oocytes were activated.6.Oocytes were cultured in CZB without glucose, sodium pyruvate and sodium lactatesupplied with different concentration of mercaptoethanol. With the concentration increasinguntil 1mM the activation rate remained more than 90%. It is obvious that mercaptoethanol can notprevent oocyte aging.7. Oocytes were cultured in CZB without glucose, sodium pyruvate and sodium lactate supplied with different concentration of dithiothreitol. With the concentration increasing until 500μM, the activation rate remained more than 90%. Furthermore, when 500μM dithiothreitol were supplied, more DOs died, and only about 20% survived, 100% of which undergone activation, hower COC did not die.8. 80% Oocytes died when they were cultured in CZB without glucose, sodium lactate, and sodium pyruvate for 24h, and all of the survived oocytes undergone spontaneous activation; When the oocytes were cultured in CZB supplied with sodium lactate 24h, all the oocytes survived and almost all of them (97%) undergone spontaneous activation, furthermore, they appeared severe abnormal and fragmental; All the oocytes cultured in CZB with sodium pyruvate remained normal and did not undergo activation.Our above results demonstrated that:1. Oocytes cultured in M199 and CZB undergo aging differently.2.Both DO and COC cultured in CZB supplied with sodium lactate or sodiumpyruvate did not undergo aging; But DO undergone aging ,however, COC not whencultured in CZB with glucose alone. This indicated that oocyte can use sodium lactate or sodium pyruvate, but utilize glucose only by cumulus cells.3. COC utilized glucose not by PPP, because of inhibiting PPP did not influence oocyte aging in CZB supplied with glucose.4. Lactate regulated oocyte aging depending on LDH ,which converted lactate to pyruvate.5.To Inhibit metablism of pyruvate in mitochondria can reduce ATP production, so it lead to oocyte aging.6. Oocyte aging can not be prevented when mercaptoethanol or DTT were supplied to CZB without glucose, lactate or pyruvate. This result indicated that pyruvate prevented oocyte aging not only by its antioxidation.7. Pyruvate can prevent oocyte aging more than lactate when they were cultured for 24h.Chapterâ…¡: the study on the effects of male on the development of mouse embryoAlthough successful embryo development is dependent upon genetic and epigenetic contributions from both the male and female, the male potential to adversely affect embryo development has been scarcely studied. It is unclear whether the sperm variation among different males would affect the outcome of oocyte evaluation by embryo development following fertilization. In the present study, variation in the developmental potential of mouse embryos was first compared between in vitro fertilization with epididymal spermatozoa from different males and Sr²⁺ parthenogenetic activation using oocytes of different qualities, and then the effect of male on fertilization and embryo development was examined using randomly chosen oocytes and spermatozoa from cauda epididymidis, vas deferens or electro-ejaculates. Rates of fertilization and blastocyst formation were significantly higher with spermatozoa from cauda epididymidis or vas deferens than with ejaculated spermatozoa. Rates of embryonic development differed significantly between different males, but not between different ejaculates of the same male. Analysis of standard errors of means and coefficients of variance indicated that as long as multiple males were involved, the variation in oocyte fertilization/activation and blastocyst formation was always higher after fertilization than after Sr²⁺ parthenogenetic activation whether spermatozoa were collected from epididymidis, vas deferens or ejaculates and regardless of oocyte qualities. It is concluded that (1) epididymal mouse spermatozoa fertilize more oocytes than ejaculated spermatozoa under identical experimental conditions; (2) like farm animals, the mice also show a remarkable male effect on the developmental potential of in vitro produced embryos although they are supposed to be less genetically diverse; (3) parthenogenetic activation is recommended for assessment of oocyte quality to exclude the effect of male.